The Role And Mechanism Of MicroRNA Regulated By X-linked Genes In The Pathophysiology Of Turner Syndrome

Turner syndrome(TS)is a female disorder caused by complete or partial absence of an X chromosome,the most typical 45,XO karyotype.It is featured by gonadal dysgenesis,short stature,congenital lymphedema,mental retardation and other systemic diseases.Ovarian dysfunction is present in most TS women and likely to be caused by rapid oocyte-loss at early fetal stages.However,the mechanisms underlying the pathogenesis of TS,especially the fetal oocyte-loss in TS,remain unclear.Meiotic-chromosome pairing error due to the loss of an X chromosome is thought to be a possible cause for TS;however,XO mice are anatomically normal and fertile,and in 45,XO patients,oogenesis is found to be arrested at very early stages even before chromosome paring is initiated,suggesting that other mechanisms might underlie the defective oogenesis in TS.In mammals,somatic cell lineages in XX embryos randomly inactivate one of their two X chromosomes,the so-called X-chromosome inactivation(XCI).As a result,the dosage of X-linked genes is compensated between XY males and XX females.Interestingly,approximately 15%X-linked genes escape XCI to variable extents.Therefore,in 45,XO where one X chromosome is missing,the XCI-escaping genes are believed to be at a lower dosage.Such haplo-insufficiency of the XCI-escaping genes has been proposed to be a cause for TS.Indeed,many XCI-escaping genes and long non-coding RNAs have been identified to be associated with TS.MicroRNAs(miRNAs)are group of small noncoding RNAs that are known to be expressed in almost all kinds of tissues and regulate a variety of developmental/physiological processes.Interestingly,distinct from Y chromosome,human X chromosome has a high density of miRNA-encoding genes.A recent study showed that X-linked miRNAs could escape meiotic XCI during spermatogenesis.Whether miRNAs are altered in 45,XO contributing to TS-associated ovary dysfunction remains unknown.The present study was carried out to compare the expression level of miRNAs between 45,XO and normal 46,XX women using genome-wide analysis and bioinformatics approaches.Since miRNAs are known to be present in the circulating systems(e.g.plasma)and their levels have been demonstrated to reflect physiological/pathological states of their donor cells from the whole body,we used circulating miRNAs from plasma for the present study.In addition,the possible regulation of TS-altered miRNAs by XCI-escaping genes and their involvement in the pathogenesis of oogenesis defects in TS were studied.Part I Plasma small RNA sequencing in 45,XO Turner syndromeObject:To detect the genome-wide differential expression of the plasma miRNAs in 45,XO Turner syndrome and 46,XX women.Materials and methods:Women with 45,XO(n=10)or 46,XX(n=10)were recruited for plasma small RNA sequencing.By the time of blood collection,all the subjects had not received hormone therapy.46,XX women with irregular menstrual cycle or reproductive endocrine diseases were excluded.We next performed bioinformatics analysis of the 85 significantly changed miRNAs in TS by Ingenuity Pathway Analysis.To verify the genome-wide analysis results,we next examined four significantly changed miRNAs with potential functions by real-time quantitative PCR(qRT-PCR)using plasma samples from another 40 TS and 40 normal women.Result(s):Total 182 circulating miRNAs were detected exhibited differential expression between in TS and normal women,and among these miRNAs,85 was shown to be significant difference(P<0.05,fold-change absolute value>1)with 40 up-regulated and 45 down-regulated.Ingenuity Pathway Analysis showed that the top pathways associated with these changed miRNAs in in each category were "Organismal injury and abnormalities","Cancer" and "Reproductive system disease".miR-486-5p,miR-320a,let-7a-5p were verified to be differential expression by real-time quantitative PCR(qRT-PCR)using plasma samples from another 40 TS and 40 normal women.Conclusion(s):There are significantly changed plasma miRNAs between 45,XO Turner syndrome and 46,XX women.Bioinformatics analysis of the significantly changed plasma miRNAs in TS are consistent with TS phenotype,including organismal abnormalities,reproductive system disease,connective tissue development and immunologlical disease,suggesting the involvement of these miRNAs in the pathogenesis of TS.Part II The relationship between differential expression miRNAs and X-chromosome inactivation escaping genesObject:To explore the relationship between differential expression miRNAs and X-chromosome inactivation escaping genesMaterials and methods:We examined the expression of miR-320a,miR-486-5p and XCI-escaping gene KDM5C,KDM6A,DDX3X from 45,XO,46,XX,46,XY and 47,XXY human peripheral blood mononuclear cells(PBMCs).Meanwhile,we verified the results in human 45,XO and 46,XX fetal gonad tissues.In vitro models,we verified the regulation between differential expression miRNAs and X-chromosome inactivation escaping genesResult(s):miR-320a,miR-486-5p are consistently upregulated in not only 45,XO plasma and peripheral blood mononuclear cells(PBMCs),but also 45,XO fetal gonad tissues;and inversely related to X chromosome numbers in PBMCs from 45,XO,46,XX,46,XY and 47,XXY human subjects.As XCI-escaping gene with X/Y chromosome homologous gene,KDM5C is lower in 45,XO PBMCs and fetal gonad tissues inversely related to miR-320a,miR-486-5p.In vitro models,except pri-miR-486-5p,pri-miR-320a,miR-320a,miR-486-5p were up-regulated after knocking down KDM5C,suggesting KDM5C inhibited miR-320a transcription.We further confirmed by luciferase reporter vectors and ChIP experiments.Knock downing H3K4me3 demethylase KDM5C,miR-320a was up-regulated,via relieving the suppression of KDM5C in miR-320a promoter.Conclusion(s):Comparing with 46,XX karyotype,miR-320a,miR-486-5p were higher expression in 45,XO PBMCs and fetal gonad tissues,which were inverse correlation of XCI escaping gene KDM5C.KDM5C suppresseed miR-320a transcription by directly binding to its promoter preventing H3K4 histone methylation Part Ⅲ The role microRNA regulated by haploinsufficient X-linked genes in gonadal dysgenesis of Turner syndromeObject:To investigate the role of miR-320a and KDM5C in gonadal dysgenesis of Turner syndromeMaterials and methods:In primary cultures of human fetal ovary(HFO)cells,we respectively transfected mimic miR-320a and KDM5C siRNA,detecting the factors known to be essential to ovary development.We further constructed Renilla luciferase reporter vectors,to verify the target of miR320a predicated by software analysis.Result(s):BMP5 and KITLG were found to be significantly decreased at mRNA level by either transfecting Mimic_miR-320a or siRNA_KDM5C into the HFO cells.Downregulation of KITLG induced by KDM5C knock-down was reversed by transfection of the miR-320a inhibitor in HFO cells.KITLG was a direct target of miR-320a.KITLG protein expression was significantly lower in 45,XO fetal gonad tissues as compared to 46,XX ones.Conclusion(s):KITLG,an essential gene for ovarian development and primordial germ cell survival,was a direct target of miR-320a.KITLG were inhibited in Mimic_miR-320a or siRNA_KDM5C.KDM5C may regulate ovarian development via targeting KITLG...